Cucurbitacin I induces immunogenic cell death and synergistically potentiates cisplatin efficacy in epithelial ovarian cancer.

[BACKGROUND] Epithelial ovarian cancer (EOC) is characterized by a suppressive tumor immune microenvironment (TIME) that undermines therapeutic efficacy. Immunogenic cell death (ICD) is a promising immunotherapy strategy attributed to ameliorating the suppressive TIME. Cisplatin (CDDP), the first-line chemotherapy for EOC, is hindered by cumulative toxicity. Cucurbitacin I (CuI), a bioactive plant-derived compound, exhibits remarkable antitumor activity in preclinical models. However, its ICD-inducing capability and cisplatin-sensitizing potential in EOC remain uninvestigated.

[PURPOSE] This study aims to systematically elucidate the ICD-inducing capability of CuI and its potential to enhance CDDP's antitumor effects in EOC both in vitro and in vivo.

[METHOD] The inhibitory effects of CuI on EOC were evaluated at the cellular level. The half-maximal inhibitory concentrations (IC) of CuI in EOC cell lines were determined via CCK-8 assay. CuI's effects on cell cycle, pyroptosis (Annexin V/PI), immune gene expression profile, and GSDME/cleaved-caspase-3 were assessed in ID8/OVCAR8 cells. Pyroptotic pathways were validated using GSDME siRNA and caspase-3 inhibitor (Z-DEVD-FMK). Following the Nomenclature Committee on Cell Death (NCCD) guidelines for ICD inducer validation, we systematically assessed damage-associated molecular patterns (DAMPs) release (CALR, ERp57, HMGB1, HSPs), dendritic cell (DC) maturation (flow cytometry analysis of murine bone marrow-derived dendritic cells (BMDCs) co-cultured with CuI-pretreated ID8 cells), and in vivo tumor vaccine potency. Intraperitoneal xenograft models evaluated CuI's antitumor efficacy and immune infiltration. Finally, CuI-CDDP synergy was tested in EOC cell lines and immunocompetent models; targets were predicted by bioinformatics (Connectivity Map and molecular docking) and validated by Surface Plasmon Resonance (SPR) to elucidate mechanistic interactions.

[RESULT] CuI induced caspase-3/GSDME-mediated pyroptosis in EOC cells, driving the release of ICD-related DAMPs, including surface-exposed CRT/ERp57, ATP, and HMGB1. Consequently, CuI-treated EOC cells promoted DC maturation, significantly increasing the proportion of CD80CD86 DCs in tumor-draining lymph nodes (from 16.07±1.02 % to 23.47±1.94 %). In ID8-luc intraperitoneal xenograft, CuI could effectively suppress ascites formation by 41.4 % and was associated with an approximately 2.8-fold increase in CD8T cell infiltration. Furthermore, the combination of CuI and CDDP synergistically enhanced pyroptosis in vitro. This synergy translated to superior antitumor effects in the ID8 xenograft model, achieving a tumor growth inhibition (TGI) of 66.02 %. This potent efficacy was accompanied by enhanced CD8T cell infiltration, a reduction in PD-1 expression on CD8T cells, and an approximately 1.78-fold increase in the splenic effector memory CD8T cell (CD44CD62L) population compared to the control. Mechanistically, CuI may enhance CDDP-induced intracellular DNA damage and reactive oxygen species accumulation by inhibiting EGFR activation.

[CONCLUSION] CuI is a novel ICD inducer that elicits caspase-3/GSDME-mediated pyroptosis and boosts antitumor immunity in EOC. Notably, it enhances the chemosensitivity of EOC to CDDP, offering a promising strategy for EOC treatment.